The overall purpose of the Scientific Cores is to provide the expertise necessary to accomplish the goals of the three projects in the program Project Grant. Since these projects share many common goals, it is more efficient to perform common experimental approaches in the Core units. Project investigators will have access to core facilities to run their own experiments and will be assisted by the core director and technical staff concerning experimental design, data interpretation and technical information. The generation of knockout mice is central to two of the proposed projects. The differentiation of wild type and targeted ES cells in vitro is also important in one project and in Core C. The establishment and execution of these procedures is labor-intensive and requires the development of reagents and technical expertise that would not make it feasible for each of the proposed projects to be undertaken individually. As a result, a knockout mouse core component is proposed to achieve the goals set by the Program/Project Grant Proposal. The gene targeting/knockout mouse core components proposed will provide technical expertise in all aspects of ES cell derivation, targeting, handling, and manipulation and all aspects of chimera production. Initially, however, this core will supervise the design and construction of targeting vector DNAs. Recent publication on ets-1 knockout mice demonstrate that expertise in morula-morula aggregation and ES cell- wild type and ES cell- tetraploid morula aggregation may be necessary for analysis (69-72, 79). The core directors expertise with embryonic lethal mutants and retrograde analysis will be useful in light of our recent discovery that our CTA-Fli targeted mutant is a recessive embryonic lethal (Project 2; New Preliminary Data). The establishment and karyotyping of ES cell lines and the maintenance of primary feeder fibroblasts, conditioned medium, and ES cell lines are required in addition to electroporation, selection, picking, and freezing of colonies. Chimeric male production includes expansion of targeted ES cell lines, superovulation of embryo donors, morula isolation and aggregation with ES cells, the generation of vasectomized males and pseudo-pregnant females, embryo transfer, and growth of chimeras to weaning. Mating of chimeric males to identify germline chimeras and generate heterozygote mutants will be the extent of core involvement.